Phospholipids with two polyunsaturated fatty acyl tails promote ferroptosis.

Phospholipids containing a single polyunsaturated fatty acyl tail (PL-PUFA1s) are considered the driving force behind ferroptosis, whereas phospholipids with diacyl-PUFA tails (PL-PUFA2s) have been rarely characterized. Dietary lipids modulate ferroptosis, but the mechanisms governing lipid metabolism and ferroptosis sensitivity are not well understood. Our research revealed a significant accumulation of diacyl-PUFA phosphatidylcholines (PC-PUFA2s) following fatty acid or phospholipid treatments, correlating with cancer cell sensitivity to ferroptosis. Depletion of PC-PUFA2s occurred in aging and Huntington's disease brain tissue, linking it to ferroptosis. Notably, PC-PUFA2s interacted with the mitochondrial electron transport chain, generating reactive oxygen species (ROS) for initiating lipid peroxidation. Mitochondria-targeted antioxidants protected cells from PC-PUFA2-induced mitochondrial ROS (mtROS), lipid peroxidation, and cell death. These findings reveal a critical role for PC-PUFA2s in controlling mitochondria homeostasis and ferroptosis in various contexts and explain the ferroptosis-modulating mechanisms of free fatty acids. PC-PUFA2s may serve as diagnostic and therapeutic targets for modulating ferroptosis.

Ferroptosis surveillance independent of GPX4 and differentially regulated by sex hormones.

Ferroptosis, a cell death process driven by iron-dependent phospholipid peroxidation, has been implicated in various diseases. There are two major surveillance mechanisms to suppress ferroptosis: one mediated by glutathione peroxidase 4 (GPX4) that catalyzes the reduction of phospholipid peroxides and the other mediated by enzymes, such as FSP1, that produce metabolites with free radical-trapping antioxidant activity. In this study, through a whole-genome CRISPR activation screen, followed by mechanistic investigation, we identified phospholipid-modifying enzymes MBOAT1 and MBOAT2 as ferroptosis suppressors. MBOAT1/2 inhibit ferroptosis by remodeling the cellular phospholipid profile, and strikingly, their ferroptosis surveillance function is independent of GPX4 or FSP1. MBOAT1 and MBOAT2 are transcriptionally upregulated by sex hormone receptors, i.e., estrogen receptor (ER) and androgen receptor (AR), respectively. A combination of ER or AR antagonist with ferroptosis induction significantly inhibited the growth of ER+ breast cancer and AR+ prostate cancer, even when tumors were resistant to single-agent hormonal therapies.

OPA1 promotes ferroptosis by augmenting mitochondrial ROS and suppressing an integrated stress response.

Ferroptosis, an iron-dependent form of nonapoptotic cell death mediated by lipid peroxidation, has been implicated in the pathogenesis of multiple diseases. Subcellular organelles play pivotal roles in the regulation of ferroptosis, but the mechanisms underlying the contributions of the mitochondria remain poorly defined. Optic atrophy 1 (OPA1) is a mitochondrial dynamin-like GTPase that controls mitochondrial morphogenesis, fusion, and energetics. Here, we report that human and mouse cells lacking OPA1 are markedly resistant to ferroptosis. Reconstitution with OPA1 mutants demonstrates that ferroptosis sensitization requires the GTPase activity but is independent of OPA1-mediated mitochondrial fusion. Mechanistically, OPA1 confers susceptibility to ferroptosis by maintaining mitochondrial homeostasis and function, which contributes both to the generation of mitochondrial lipid reactive oxygen species (ROS) and suppression of an ATF4-mediated integrated stress response. Together, these results identify an OPA1-controlled mitochondrial axis of ferroptosis regulation and provide mechanistic insights for therapeutically manipulating this form of cell death in diseases.

A cell state-specific metabolic vulnerability to GPX4-dependent ferroptosis in glioblastoma.

Glioma cells hijack developmental programs to control cell state. Here, we uncover a glioma cell state-specific metabolic liability that can be therapeutically targeted. To model cell conditions at brain tumor inception, we generated genetically engineered murine gliomas, with deletion of p53 alone (p53) or with constitutively active Notch signaling (N1IC), a pathway critical in controlling astrocyte differentiation during brain development. N1IC tumors harbored quiescent astrocyte-like transformed cell populations while p53 tumors were predominantly comprised of proliferating progenitor-like cell states. Further, N1IC transformed cells exhibited increased mitochondrial lipid peroxidation, high ROS production and depletion of reduced glutathione. This altered mitochondrial phenotype rendered the astrocyte-like, quiescent populations more sensitive to pharmacologic or genetic inhibition of the lipid hydroperoxidase GPX4 and induction of ferroptosis. Treatment of patient-derived early-passage cell lines and glioma slice cultures generated from surgical samples with a GPX4 inhibitor induced selective depletion of quiescent astrocyte-like glioma cell populations with similar metabolic profiles. Collectively, these findings reveal a specific therapeutic vulnerability to ferroptosis linked to mitochondrial redox imbalance in a subpopulation of quiescent astrocyte-like glioma cells resistant to standard forms of treatment.

MDM2 and MDMX promote ferroptosis by PPARα-mediated lipid remodeling.

MDM2 and MDMX, negative regulators of the tumor suppressor p53, can work separately and as a heteromeric complex to restrain p53's functions. MDM2 also has pro-oncogenic roles in cells, tissues, and animals that are independent of p53. There is less information available about p53-independent roles of MDMX or the MDM2-MDMX complex. We found that MDM2 and MDMX facilitate ferroptosis in cells with or without p53. Using small molecules, RNA interference reagents, and mutant forms of MDMX, we found that MDM2 and MDMX, likely working in part as a complex, normally facilitate ferroptotic death. We observed that MDM2 and MDMX alter the lipid profile of cells to favor ferroptosis. Inhibition of MDM2 or MDMX leads to increased levels of FSP1 protein and a consequent increase in the levels of coenzyme Q10, an endogenous lipophilic antioxidant. This suggests that MDM2 and MDMX normally prevent cells from mounting an adequate defense against lipid peroxidation and thereby promote ferroptosis. Moreover, we found that PPARα activity is essential for MDM2 and MDMX to promote ferroptosis, suggesting that the MDM2-MDMX complex regulates lipids through altering PPARα activity. These findings reveal the complexity of cellular responses to MDM2 and MDMX and suggest that MDM2-MDMX inhibition might be useful for preventing degenerative diseases involving ferroptosis. Furthermore, they suggest that MDM2/MDMX amplification may predict sensitivity of some cancers to ferroptosis inducers.

Identification of essential sites of lipid peroxidation in ferroptosis.

Ferroptosis, an iron-dependent form of cell death driven by lipid peroxidation, provides a potential treatment avenue for drug-resistant cancers and may play a role in the pathology of some degenerative diseases. Identifying the subcellular membranes essential for ferroptosis and the sequence of their peroxidation will illuminate drug discovery strategies and ferroptosis-relevant disease mechanisms. In this study, we employed fluorescence and stimulated Raman scattering imaging to examine the structure-activity-distribution relationship of ferroptosis-modulating compounds. We found that, although lipid peroxidation in various subcellular membranes can induce ferroptosis, the endoplasmic reticulum (ER) membrane is a key site of lipid peroxidation. Our results suggest an ordered progression model of membrane peroxidation during ferroptosis that accumulates initially in the ER membrane and later in the plasma membrane. Thus, the design of ER-targeted inhibitors and inducers of ferroptosis may be used to optimally control the dynamics of lipid peroxidation in cells undergoing ferroptosis.

Red blood cell exposure increases chondrocyte susceptibility to oxidative stress following hemarthrosis.

OBJECTIVE: The detrimental effects of blood exposure on articular tissues are well characterized, but the individual contributions of specific whole blood components are yet to be fully elucidated. Better understanding of mechanisms that drive cell and tissue damage in hemophilic arthropathy will inform novel therapeutic strategies. The studies here aimed to identify the specific contributions of intact and lysed red blood cells (RBCs) on cartilage and the therapeutic potential of Ferrostatin-1 in the context of lipid changes, oxidative stress, and ferroptosis. METHODS: Changes to biochemical and mechanical properties following intact RBC treatment were assessed in human chondrocyte-based tissue-engineered cartilage constructs and validated against human cartilage explants. Chondrocyte monolayers were assayed for changes to intracellular lipid profiles and the presence of oxidative and ferroptotic mechanisms. RESULTS: Markers of tissue breakdown were observed in cartilage constructs without parallel losses in DNA (control: 786.3 (102.2) ng/mg; RBCINT: 751 (126.4) ng/mg; P = 0.6279), implicating nonlethal chondrocyte responses to intact RBCs. Dose-dependent loss of viability in response to intact and lysed RBCs was observed in chondrocyte monolayers, with greater toxicity observed with lysates. Intact RBCs induced changes to chondrocyte lipid profiles, upregulating highly oxidizable fatty acids (e.g., FA 18:2) and matrix disrupting ceramides. RBC lysates induced cell death via oxidative mechanisms that resemble ferroptosis. CONCLUSIONS: Intact RBCs induce intracellular phenotypic changes to chondrocytes that increase vulnerability to tissue damage while lysed RBCs have a more direct influence on chondrocyte death by mechanisms that are representative of ferroptosis.

Comparison of the Cannabinoid and Terpene Profiles in Commercial Cannabis from Natural and Artificial Cultivation.

Interest in cultivating cannabis for medical and recreational purposes is increasing due to a dramatic shift in cannabis legislation worldwide. Therefore, a comprehensive understanding of the composition of secondary metabolites, cannabinoids, and terpenes grown in different environmental conditions is of primary importance for the medical and recreational use of cannabis. We compared the terpene and cannabinoid profiles using gas/liquid chromatography and mass spectrometry for commercial cannabis from genetically identical plants grown indoors using artificial light and artificially grown media or outdoors grown in living soil and natural sunlight. By analyzing the cannabinoids, we found significant variations in the metabolomic profile of cannabis for the different environments. Overall, for both cultivars, there were significantly greater oxidized and degraded cannabinoids in the indoor-grown samples. Moreover, the outdoor-grown samples had significantly more unusual cannabinoids, such as C4- and C6-THCA. There were also significant differences in the terpene profiles between indoor- and outdoor-grown cannabis. The outdoor samples had a greater preponderance of sesquiterpenes including ß-caryophyllene, α-humulene, α-bergamotene, α-guaiene, and germacrene B relative to the indoor samples.

Application of Ion Mobility Mass Spectrometry in Lipidomics.

Lipids play significant roles in biological system, and the study of lipid metabolisms may provide a new insight into the diagnosis and pathophysiology of diseases. Recent developments in high-resolution mass spectrometry techniques combined with high-performance chromatographic methods provide deep insight into lipid analysis. Addition of ion mobility mass spectrometry orthogonal to LC-MS analysis workflow enhances separation of complex lipids, improve isomers resolution, and intensify confidence in lipid identification and characterization. In this chapter, we describe the principle of travelling wave ion mobility mass spectrometry (TWIMS) and its applications in untargeted LC-MS analysis for characterizing the structural diversity and complexity of lipid species in biological samples.

Liver metabolomics reveals increased oxidative stress and fibrogenic potential in gfrp transgenic mice in response to ionizing radiation.

Although radiation-induced tissue-specific injury is well documented, the underlying molecular changes resulting in organ dysfunction and the consequences thereof on overall metabolism and physiology have not been elucidated. We previously reported the generation and characterization of a transgenic mouse strain that ubiquitously overexpresses Gfrp (GTPH-1 feedback regulatory protein) and exhibits higher oxidative stress, which is a possible result of decreased tetrahydrobiopterin (BH4) bioavailability. In this study, we report genotype-dependent changes in the metabolic profiles of liver tissue after exposure to nonlethal doses of ionizing radiation. Using a combination of untargeted and targeted quantitative mass spectrometry, we report significant accumulation of metabolites associated with oxidative stress, as well as the dysregulation of lipid metabolism in transgenic mice after radiation exposure. The radiation stress seems to exacerbate lipid peroxidation and also results in higher expression of genes that facilitate liver fibrosis, in a manner that is dependent on the genetic background and post-irradiation time interval. These findings suggest the significance of Gfrp in regulating redox homeostasis in response to stress induced by ionizing radiation affecting overall physiology.

Evaluating the Ability of External Electric Fields to Accelerate Reactions in Solution.

This study investigates the catalytic effects of external electric fields (EEFs) on two reactions in solution: the Menshutkin reaction and the Chapman rearrangement. Utilizing a scanning tunneling microscope-based break-junction (STM-BJ) setup and monitoring reaction rates through high-performance liquid chromatography connected to a UV detector (HPLC-UV) and ultraperformance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-q-ToF-MS), we observed no rate enhancement for either reaction under ambient conditions. Density functional theory (DFT) calculations indicate that electric field-induced changes in reactant orientation and the minimization of activation energy are crucial factors in determining the efficacy of EEF-driven catalysis. Our findings suggest that the current experimental setups and field strengths are insufficient to catalyze these reactions, underscoring the importance of these criteria in assessing the reaction candidates.

Fatal COVID-19 pulmonary disease involves ferroptosis.

SARS-CoV-2 infection causes severe pulmonary manifestations, with poorly understood mechanisms and limited treatment options. Hyperferritinemia and disrupted lung iron homeostasis in COVID-19 patients imply that ferroptosis, an iron-dependent cell death, may occur. Immunostaining and lipidomic analysis in COVID-19 lung autopsies reveal increases in ferroptosis markers, including transferrin receptor 1 and malondialdehyde accumulation in fatal cases. COVID-19 lungs display dysregulation of lipids involved in metabolism and ferroptosis. We find increased ferritin light chain associated with severe COVID-19 lung pathology. Iron overload promotes ferroptosis in both primary cells and cancerous lung epithelial cells. In addition, ferroptosis markers strongly correlate with lung injury severity in a COVID-19 lung disease model using male Syrian hamsters. These results reveal a role for ferroptosis in COVID-19 pulmonary disease; pharmacological ferroptosis inhibition may serve as an adjuvant therapy to prevent lung damage during SARS-CoV-2 infection.

Multimodal mass spectrometry imaging identifies cell-type-specific metabolic and lipidomic variation in the mammalian liver.

Spatial single-cell omics provides a readout of biochemical processes. It is challenging to capture the transient lipidome/metabolome from cells in a native tissue environment. We employed water gas cluster ion beam secondary ion mass spectrometry imaging ([H2O]n>28K-GCIB-SIMS) at ≤3 µm resolution using a cryogenic imaging workflow. This allowed multiple biomolecular imaging modes on the near-native-state liver at single-cell resolution. Our workflow utilizes desorption electrospray ionization (DESI) to build a reference map of metabolic heterogeneity and zonation across liver functional units at tissue level. Cryogenic dual-SIMS integrated metabolomics, lipidomics, and proteomics in the same liver lobules at single-cell level, characterizing the cellular landscape and metabolic states in different cell types. Lipids and metabolites classified liver metabolic zones, cell types and subtypes, highlighting the power of spatial multi-omics at high spatial resolution for understanding celluar and biomolecular organizations in the mammalian liver.

Ferroptosis inhibition by oleic acid mitigates iron-overload-induced injury.

Iron overload, characterized by accumulation of iron in tissues, induces a multiorgan toxicity whose mechanisms are not fully understood. Using cultured cell lines, Caenorhabditis elegans, and mice, we found that ferroptosis occurs in the context of iron-overload-mediated damage. Exogenous oleic acid protected against iron-overload-toxicity in cell culture and Caenorhabditis elegans by suppressing ferroptosis. In mice, oleic acid protected against FAC-induced liver lipid peroxidation and damage. Oleic acid changed the cellular lipid composition, characterized by decreased levels of polyunsaturated fatty acyl phospholipids and decreased levels of ether-linked phospholipids. The protective effect of oleic acid in cells was attenuated by GW6471 (PPAR-α antagonist), as well as in Caenorhabditis elegans lacking the nuclear hormone receptor NHR-49 (a PPAR-α functional homologue). These results highlight ferroptosis as a driver of iron-overload-mediated damage, which is inhibited by oleic acid. This monounsaturated fatty acid represents a potential therapeutic approach to mitigating organ damage in iron overload individuals.

Proteomic analysis of ferroptosis pathways reveals a role of CEPT1 in suppressing ferroptosis.

Ferroptosis has been recognized as a unique cell death modality driven by excessive lipid peroxidation and unbalanced cellular metabolism. In this study, we established a protein interaction landscape for ferroptosis pathways through proteomic analyses, and identified choline/ethanolamine phosphotransferase 1 (CEPT1) as a lysophosphatidylcholine acyltransferase 3 (LPCAT3)-interacting protein that regulates LPCAT3 protein stability. In contrast to its known role in promoting phospholipid synthesis, we showed that CEPT1 suppresses ferroptosis potentially by interacting with phospholipases and breaking down certain pro-ferroptotic polyunsaturated fatty acid (PUFA)-containing phospholipids. Together, our study reveals a previously unrecognized role of CEPT1 in suppressing ferroptosis.

Targeted delivery of napabucasin with radiotherapy improves outcomes in diffuse midline glioma.

BACKGROUND: Diffuse midline glioma (DMG) is the most aggressive primary brain tumor in children. All previous studies examining the role of systemic agents have failed to demonstrate a survival benefit; the only standard of care is radiation therapy (RT). Successful implementation of radiosensitization strategies in DMG remains an essential and promising avenue of investigation. We explore the use of Napabucasin, an NAD(P)H quinone dehydrogenase 1 (NQO1)-bioactivatable reactive oxygen species (ROS)-inducer, as a potential therapeutic radiosensitizer in DMG. METHODS: In this study, we conduct in vitro and in vivo assays using patient-derived DMG cultures to elucidate the mechanism of action of Napabucasin and its radiosensitizing properties. As penetration of systemic therapy through the blood-brain barrier (BBB) is a significant limitation to the success of DMG therapies, we explore focused ultrasound (FUS) and convection-enhanced delivery (CED) to overcome the BBB and maximize therapeutic efficacy. RESULTS: Napabucasin is a potent ROS-inducer and radiosensitizer in DMG, and treatment-mediated ROS production and cytotoxicity are dependent on NQO1. In subcutaneous xenograft models, combination therapy with RT improves local control. After optimizing targeted drug delivery using CED in an orthotopic mouse model, we establish the novel feasibility and survival benefit of CED of Napabucasin concurrent with RT. CONCLUSIONS: As nearly all DMG patients will receive RT as part of their treatment course, our validation of the efficacy of radiosensitizing therapy using CED to prolong survival in DMG opens the door for exciting novel studies of alternative radiosensitization strategies in this devastating disease while overcoming limitations of the BBB.

Engineering tumor-colonizing E. coli Nissle 1917 for detection and treatment of colorectal neoplasia.

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.

DISC-3D: dual-hydrogel system enhances optical imaging and enables correlative mass spectrometry imaging of invading multicellular tumor spheroids.

Multicellular tumor spheroids embedded in collagen I matrices are common in vitro systems for the study of solid tumors that reflect the physiological environment and complexities of the in vivo environment. While collagen I environments are physiologically relevant and permissive of cell invasion, studying spheroids in such hydrogels presents challenges to key analytical assays and to a wide array of imaging modalities. While this is largely due to the thickness of the 3D hydrogels that in other samples can typically be overcome by sectioning, because of their highly porous nature, collagen I hydrogels are very challenging to section, especially in a manner that preserves the hydrogel network including cell invasion patterns. Here, we describe a novel method for preparing and cryosectioning invasive spheroids in a two-component (collagen I and gelatin) matrix, a technique we term dual-hydrogel in vitro spheroid cryosectioning of three-dimensional samples (DISC-3D). DISC-3D does not require cell fixation, preserves the architecture of invasive spheroids and their surroundings, eliminates imaging challenges, and allows for use of techniques that have infrequently been applied in three-dimensional spheroid analysis, including super-resolution microscopy and mass spectrometry imaging.

Regulation of VKORC1L1 is critical for p53-mediated tumor suppression through vitamin K metabolism.

Here, we identified vitamin K epoxide reductase complex subunit 1 like 1 (VKORC1L1) as a potent ferroptosis repressor. VKORC1L1 protects cells from ferroptosis by generating the reduced form of vitamin K, a potent radical-trapping antioxidant, to counteract phospholipid peroxides independent of the canonical GSH/GPX4 mechanism. Notably, we found that VKORC1L1 is also a direct transcriptional target of p53. Activation of p53 induces downregulation of VKORC1L1 expression, thus sensitizing cells to ferroptosis for tumor suppression. Interestingly, a small molecular inhibitor of VKORC1L1, warfarin, is widely prescribed as an FDA-approved anticoagulant drug. Moreover, warfarin represses tumor growth by promoting ferroptosis in both immunodeficient and immunocompetent mouse models. Thus, by downregulating VKORC1L1, p53 executes the tumor suppression function by activating an important ferroptosis pathway involved in vitamin K metabolism. Our study also reveals that warfarin is a potential repurposing drug in cancer therapy, particularly for tumors with high levels of VKORC1L1 expression.

Dietary restriction of cysteine and methionine sensitizes gliomas to ferroptosis and induces alterations in energetic metabolism.

Ferroptosis is mediated by lipid peroxidation of phospholipids containing polyunsaturated fatty acyl moieties. Glutathione, the key cellular antioxidant capable of inhibiting lipid peroxidation via the activity of the enzyme glutathione peroxidase 4 (GPX-4), is generated directly from the sulfur-containing amino acid cysteine, and indirectly from methionine via the transsulfuration pathway. Herein we show that cysteine and methionine deprivation (CMD) can synergize with the GPX4 inhibitor RSL3 to increase ferroptotic cell death and lipid peroxidation in both murine and human glioma cell lines and in ex vivo organotypic slice cultures. We also show that a cysteine-depleted, methionine-restricted diet can improve therapeutic response to RSL3 and prolong survival in a syngeneic orthotopic murine glioma model. Finally, this CMD diet leads to profound in vivo metabolomic, proteomic and lipidomic alterations, highlighting the potential for improving the efficacy of ferroptotic therapies in glioma treatment with a non-invasive dietary modification.